Bilirubin is a degradation product of hemoglobin. Approximately 200 to 230 mg of bilirubin and its derivatives are formed each day in the normal human adult. As part of normal human metabolic processes, the major portion of this daily bilirubin production is excreted or degraded into other derivatives.
Excessive amounts of bilirubin occur within the human body through overproduction of bilirubin as in the case of excessive hemolysis or by retention of bilirubin due, for example, to liver failure. The result of excessive bilirubin within the human body is jaundice. Jaundice is characterized by markedly elevated serum bilirubin levels, for example, 10 mg of bilirubin per dL of serum or higher compared with the normal adult range of 0.1 to about 1 mg of bilirubin per dL of serum. There is increasing evidence that excessive amounts of bilirubin in the blood lead to an undesirable increase in bilirubin concentration within body cells which interferes with various cellular processes. Given this background, the clinical diagnostic significance of bilirubin, in tests for liver and other related organ functions, is self evident.
Perhaps the most widely used assay for bilirubin has been the so called diazo method. In this method, a sample of liquid suspected of containing bilirubin is contacted with a reagent composition which includes a diazonium salt. The diazonium salt reacts with bilirubin to form two azobilirubin fragments. The azobilirubin has an extinction coefficient which is higher than that of bilirubin itself and is therefore easily detectable.
Many diazonium salts have been suggested for use in the diazo method for determining bilirubin. For example, certain 2,4- and 2,5-phenyldiazonium salts (e.g. 2,4- and 2,5-dichlorophenyldiazonium salts) and diazotized sulfanilamide have been used for the detection of bilirubin in serum and urine. However, methods using these diazonium salts are known to be relatively insensitive. Further, some of these diazonium salts, when dry, are explosively unstable, i.e. subject to shock induced decomposition. Thus, handling of these compounds in bilirubin assays, and particularly dry assays, is quite hazardous.
Certain substituted sulfanilamide and carbonamide diazonium salts which are less prone to shock induced decomposition have been found useful in bilirubin assays. These salts and assays are the subject of commonly assigned and copending U.S. Ser. No. 344,433, filed February 2, 1982 by our colleagues, B. E. Babb and G. M. Dappen, and entitled DIAZONIUM SALT FOR BILIRUBIN ASSAY now U.S. Pat. No. 4,468,467. Those salts and assays represent a significant improvement in the clinical chemistry art, overcoming the shortcomings of previously-known bilirubin assays. This improved assay is also described by Babb and co-authors in Clin. Chem., 29(1), pp. 37-41 (1983).
However, there is a need to provide further improvements in the bilirubin assays described and claimed in aforementioned U.S. Pat. No. 4,468,467. With a small percentage of patient serum samples, e.g. those obtained from hemodialysis or other renal-defective patients, interferences were observed to be influential in the end result, detracting from assay accuracy. It is desirable to remove such interferences, thereby providing an assay that is highly accurate with all patient samples including samples obtained from patients having kidney problems.
Known procedures for eliminating interferences in assays include sample pretreatment, sample blanking and polychromatic (i.e. multiple wavelength) analyses. Each of these procedures, however, has its disadvantages. Sample pretreatment is a tedious and imprecise operation and is not readily adaptable to dry chemistry assays. Sample blanking doubles the effort, sample size and cost of each assay and may cause a decrease in precision. The known polychromatic analysis requires pure standards and knowledge of the exact molecular identity or concentrations of predetermined interferents. See, e.g. Hahn et al, Clin. Chem., 25(6), pp. 951-959 (1979).
None of these known procedures has proved effective for eliminating the observed interference in the bilirubin assay described and claimed in U.S. Pat. No. 4,468,467 noted hereinabove. Neither the identity of the interferent nor its concentration (which can vary from sample to sample) is known. This precludes use of the polychromatic analysis which requires knowledge of the interferent or its concentration. The other procedures are equally useless in this instance.
Therefore, there is a need in the art for a diazo bilirubin assay having improved accuracy for all patient samples, which assay overcomes the effect of an undetermined interferent or where the interferent is formed in situ, i.e. during the analysis.